There is considerable interest in cell-based therapies for the repair and tissue regeneration of myocardial tissue. Embryonic stem cells are explored as an unlimited source of cells for cardiac repair, as they can differentiate into cardiomyocytes in culture. A better understanding of the regulation of this differentiation process would greatly aid in the generation of cardiomyocytes, and may lead to methods to improve the yield, control the quality and homogeneity, and direct subtype characteristics of the cardiomyocytes. Little is known about the signaling pathways and transcription factors that control the differentiation potential and cardiomyocyte differentiation of human embryonic stem cells. Oct4, Sox2 and Nanog function as essential transcription factors for self-renewal and pluripotency, yet may also play key roles in the initiation of differentiation into cardiomyocytes. Signaling by TGF-2 family proteins controls self-renewal, pluripotency and differentiation of stem cells, and autocrine signaling by TGF-2 family proteins is likely to play a key role in the self-renewal and differentiation of embryonic stem cells. TGF-2 family proteins exert gene expression responses through Smads, which enhance or repress the transcription activities of transcription factors at target genes, and thus function as cell-intrinsic mediators of differentiation. Through this mode of action, Smads are likely to regulate the expression levels and functions of embryonic stem cell transcription factors, such as Oct4, Sox2 and Nanog, and regulate the selection and progression of differentiation of cardiomyocytes from embryonic stem cells. The overall goals of this proposal are to (1) evaluate the regulation of expression and activities of the Oct4, Sox2 and Nanog by TGF-2 family/Smad signaling, (2) correlate this level of control with the differentiation potential and characteristics of differentiation along the cardiomyocyte lineage, (3) to use this knowledge to generate cardiomyocyte progenitors with high efficiency and defined characteristics. We hypothesize that (1) Smad signaling by TGF-2 family proteins regulates the expression and activities of the embryonic stem cell transcription factors, (2) alterations in Smad signaling and Oct4, Sox2 and/or Nanog activities modify the differentiation capacity of the cells and specifically cardiomyocyte differentiation. We propose three Aims: (1) to examine the regulation of embryonic stem cell transcription factor expression and activities by TGF-2 family signaling and to correlate these findings with cardiomyocyte lineage differentiation, (2) to study the roles of individual Smads in the regulation of Oct4, Sox2 and Nanog, and the roles of these Smads and Oct4, Sox2 and Nanog themselves in cardiomyocyte lineage differentiation, (3) To examine the in vivo differentiation and tissue integration characteristics of cardiomyocyte precursors derived from embryonic stem cells following manipulations that favor the generation of these cells in culture. PUBLIC HEALTH RELEVANCE: Cell-based therapies for the repair and tissue regeneration of heart tissue, for example following heart infarct, may provide great promise, and human embryonic stem cells are being considered as a cello source for these cells, called cardiomyocytes. We now propose a research plan in which we try to understand signaling pathways that may direct the differentiation of cardiomyocytes. We will explore how to modify these pathways and hope to design approaches that increase and improve the generation and characteristics of the cardiomyocytes. This will be tested through a combination of cell culture experiments and a new method in which the cardiomyocytes are injected into the heart muscle of mice with a heart infarct.